Summary of Work: Mass spectrometry is playing a significant role in the identification of unknown proteins at trace levels, in the identification of posttranslational modifications of proteins, and in the identification of proteins that are part of functional/disease related biological complexes. This information can be vital to understanding signal transduction pathways and the regulation of function of the involved proteins. Critical parts of this project are to develop procedures for handling and isolation of sub-picomole levels of proteins, specifically modified proteins (e.g., phosphorylated), intact protein complexes, and their digests in a manner compatible with the final instrumental determinations. Specific aims:1.Identification of proteins involved in inhibited spermiation.2.Determine sites of phosphorylation of tristetraprolin, a regulator of TNFa, a major mediator of inflammatory response.3.Characterize the regulatory domain of PP5 and its site of interaction.4.Determination the structural basis of the inhibition of BKca channels by phosphokinases.5.Elucidation of phosphorylation sites on p53 and of the interrelationship between phosphorylation of specific sites and biological activity.Biological processes often involve specific non-covalent and/or covalent interactions. Understanding the structures of functional intermediates and products is important in determining biochemical mechanisms and how specific molecules interact with endogenous compounds. Specific aims:1.Identify the amino acids of b-polymerase that can form the covalent intermediate with the abasic site in excision-repair.2.Develop the mass spectrometric capability to characterize specific, higher- order non-covalent, heterogeneous complexes, for example GCN4 peptides and dsDNA. - mass spectrometry, electrospray ionization tandem mass spectrometry non-covalent complexes phosphorylation p53